Process for the preparation of amides

ABSTRACT

A novel biotechnological process for the preparation of nitriles, starting from amides, is described. Micro-organisms of the genus Amycolatopsis, Actinomadura or Rhodococcus are employed for this process.

[0001] The invention relates to novel microorganisms of the genusActinomadura, Amycolatopsis or Rhodococcus, and to a novel process forthe preparation of amides using these microorganisms or using enzymeextracts of these microorganisms.

[0002] For amides such as, for example, nicotinamide, a vitamin of thevitamin B complex which is essential to animals and man, a number ofbiotechnological processes are already known. Generally, it is knownthat microorganisms containing nitrile hydratase convert nitriles to thecorresponding amides. Thus EP-A-0 188 316 describes a process for thepreparation of nicotinamide starting from 3-cyanopyridine usingmicroorganisms of the genus Rhodococcus, Arthrobacter or Microbacterium.

[0003] A disadvantage of this process is that these micro-organisms haveonly a low activity for the conversion of 3-cyanopyridine tonicotinamide.

[0004] EP-A-0 307 926 describes the conversion of 3-cyano-pyridine tonicotinamide by means of microorganisms of the species Rhodococcusrhodochrous J1. In order that these microorganisms catalyse the desiredconversion, they must be induced.

[0005] A further disadvantage of this process is that Rhodococcusrhodochrous J1 is red-coloured and accordingly a discoloration of theproduct takes place. In addition, this microorganism has a low heatstability and is inhibited, for example, by the substrate3-cyano-pyridine.

[0006] A further process for the preparation of nicotinamide startingfrom 3-cyanopyridine by means of microorganisms of the speciesRhodococcus rhodochrous J1 is described in EP-A-0 362 829. In order toincrease the specific activity of the microorganisms containing nitrilehydratase, urea or a urea derivative was added to the culturing mediumas an inducer. As in the process described beforehand, a discolorationof the product also takes place in this process.

[0007] In addition, WO 95/17 505 describes a process for the preparationof aromatic amides starting from the corresponding nitriles by means ofmicroorganisms of the species Rhodococcus rhodochrous M33. Adisadvantage of this process is the red colouration of Rhodococcusrhodochrous M33 and also the high K_(M) value for the substrate3-cyanopyridine.

[0008] The object of the present invention was to eliminate thesedisadvantages and to make available a process for the preparation ofamides in which the corresponding amides can be isolated in good yieldand purity.

[0009] This object is achieved by the novel microorganisms according toclaims 1 and 3, and by the process according to claim 6.

[0010] According to the invention, the process is carried out byconverting a nitrile, as substrate, to the corresponding amide by meansof microorganisms of the genus Actinomadura, Amycolatopsis orRhodococcus, using an enzyme extract of these microorganisms or by meansof purified nitrile hydratase of microorganisms of the genusAmycolatopsis or Actinomadura.

[0011] The nitriles employed for the biotransformation such as, forexample, 3-cyanopyridine are commercially available compounds.

[0012] The microorganisms according to the invention are able to convertnitriles as substrates into the corresponding amides. Preferably, thesemicroorganisms have the ability to grow on nitriles or amides as thesole C and/or N source.

[0013] The microorganisms according to the invention are obtainable bymeans of suitable selection, for example, from soil samples, sludge orwaste water with the aid of customary microbiological techniques.Expediently, the microorganisms are selected by growth with nitriles oramides as the preferably sole C and N source in the presence of cobaltions. Nitriles and amides suitable for selection are, in particular, thenitriles also employed as substrates in the later biotransformation andthe corresponding amides obtainable therefrom. Suitable growth media arelikewise known to the person skilled in the art, for example the mediumdescribed in Table 1 can be used.

[0014] Customarily, the microorganisms are cultured (grown) in the samemanner even before the actual biotransformation, the abovementionedmedia being used.

[0015] As known professionally, a nitrile hydratase is only formed whenthe growth medium contains cobalt ions as a cofactor. Suitable “cobaltcompounds generating cobalt ions” are Co²⁺ or Co³⁺ salts. Examples ofCo²⁺ and Co³⁺ salts are cobalt chlorides, cobalt sulphates and cobaltacetates.

[0016] Expediently, the cobalt compound employed is a Co²⁺ salt such as,for example, CoCl₂. Growth, however, can also be carried out in thepresence of vitamin B12 together with metallic cobalt or other cobaltcompounds which generate a cobalt ion in situ. Expediently, the cobaltcompound is employed in an amount from 1 to 10 mg/l, preferably from 1to 3 mg/l.

[0017] Customarily, growth is carried out at a temperature from 20 to50° C. and at a pH between pH 5 and pH 8, preferably from 30 to 45° C.and between pH 5.5 and pH 7.5.

[0018] The actual biotransformation can be carried out usingmicroorganisms of the genus Actinomadura, Amycolatopsis, using an enzymeextract of these microorganisms or by means of purified nitrilehydratase from these microorganisms. Expediently, the biotransformationis carried out using microorganisms of the species Actinomadura spadix,for example the isolates Actinomadura spadix E3733, Actinomadura spadixE3736, Actinomadura spadix 45A32, Actinomadura spadix 4501 orActinomadura spadix C15. The biotransformation is preferably carried outusing microorganisms corresponding to the species Amycolatopsis NE 31and Amycolatopsis NA40 or their functionally equivalent variants andmutants. Micro-organisms corresponding to the species Amycolatopsis NA40are particularly preferably employed. Microorganisms of the speciesmentioned were deposited on 03.06.1997 in the Deutschen Sammlung vonMikroorganismen und Zellkulturen GmbH [German Collection ofMicroorganisms and Cell Cultures GmbH], Mascheroderweg 1b, D-38124Brunswick under the designations Amycolatopsis NE 31 and AmycolatopsisNA40 according to the Budapest Convention and have the deposit numbersDSMZ 11616 and DSMZ 11617 respectively. These two microorganisms havebeen more accurately identified and are to be assigned to species of thegenus Amycolatopsis which have not yet been described in the literature.

[0019] Accordingly, the invention also relates to microorganisms of thegenus Amycolatopsis or Actinomadura which are capable of converting anamide into a nitrile, in particular microorganisms with the designationAmycolatopsis NA40 (DSMZ 11617) and Amycolatopsis NE31 (DSMZ 11616).

[0020] In addition, it has been found that specific microorganisms ofthe genus Rhodococcus have better properties for the conversion ofnitriles to amides than the Rhodococcus rhodochrous J1 described inEP-A-0 362 829. These microorganisms are Rhodococcus GF674, RhodococcusGF578, Rhodococcus GF473, Rhodococcus GF270 (DSMZ 12211) and RhodococcusGF376 (DSMZ 12175) or their functionally equivalent variants andmutants. The microorganism DSMZ 12175 was deposited on 15.5.1998 and themicroorganism DSMZ 12211 on 8.6.1998 in the Deutschen Sannlung vonMikroorganismen und Zellkulturen GmbH [German Collection ofMicroorganisms and Cell Cultures GmbH] according to the BudapestConvention.

[0021] The Rhodococcus strains GF270, GF376, GF473, GF578 and GF674 havebeen assigned according to identification to species of the genusRhodococcus which are not yet described in the literature. Accordingly,the invention also relates to the microorganisms Rhodococcus GF270,Rhodococcus GF376, Rhodococcus GF473, Rhodococcus GF578 and RhodococcusGF674.

[0022] Unlike the microorganisms of the genus Actinomadura orAmycolatopsis, the microorganisms of the genus Rhodococcus areexpediently induced before the actual conversion. Suitable inducers arethose described in EP-A-0 307 926, such as, for example, acetamide,butyramide, methacrylamide, propionamide, crotonamide and valeramide.

[0023] “Functionally equivalent variants and mutants” is understood asmeaning microorganisms which are derived from the abovementioned sourceorganisms and essentially have the same characteristics and functions asthese. Variants and mutants of this type can be formed by chance, e.g.by UV irradiation or by mutagenic chemicals. Identification ofAmycolatopsis NA40 Colour of aerial mycelium white Colour of substratemycelium orange Colour of diffused pigment − Sugar spectrum ARA + GAL +MAD − XYL − GLU tr RIB + Type A DAP DL Menaquinones (in %) 8/4 − 9/0 (+)9/2 + 9/4 +++ 9/6 − 9/8 − 16S rDNA homology 96.9% Phospholipids such asnot investigated PE, OH-PE, lyso PE, met PE, PC, NPG, PI, PIM, PG, DPG,GL Fatty acids iso 16 +++ iso 15 + iso 17 (+) anteiso 15 (+) anteiso 17(+) 10-Me 16 − 10-Me 17 + 2-OH 15 + 2-OH 16 + Type 3f MS −Identification of Amycolatopsis NE31 Colour of aerial mycelium whiteColour of substrate mycelium orange Colour of diffused pigment − Sugarspectrum ARA + GAL + MAD − XYL − GLU tr RIB + Type A DAP DL Menaquinones(in %) 8/4 − 9/0 (+) 9/2 + 9/4 +++ 9/6 − 9/8 − 16S rDNA homology 96.1%Phospholipids such as not investigated PE, OH-PE, lyso PE, met PE, PC,NPG, PI, PIM, PG DPG, GL Fatty acids iso 16 +++ iso 15 + iso 17 (+)anteiso 15 (+) anteiso 17 (+) 10-Me 16 − 10-Me 17 + 2-OH 15 + 2-OH 16 +Type 3f MS − Abbreviations and explanations for the identification (+)1-5% + 5-15% ++ 15-30% +++ >30% DAP diaminopimelic acid ARA arabinoseGAL galactose MAD madurose XYL xylose GLU glucose RIB ribose Sugar typesaccording to Lechevalier et al. 1971 Fatty acid types according toKroppenstedt 1985 and 1992. 9/4 MK-9 (H₄) 9/6 MK-9 (H₆) 9/8 MK-9 (H₈) MSmycolic acids PE phosphatidylethanolamine OH-PE hydroxy-PE met PEphosphatidimethylethanol- amine PC phosphatidylcholine NPGphosphatidylglucosamine PI phosphatidylinositol PIM phosphatidylinositolmannoside PG phosphatidylglycerol DPG diphosphatidylglycerol GLglycolipids Fatty acids iso-16 isohexadecanoic acids or14-methylpentadecanoic acids 10-Me-18 tuberculostearic acid 2-OH-162-hydroxypalmitic acid

[0024] Identification of GF270, GF376, GF473, GF578 and GF674 Theidentification of these strains is based on 5 characteristics which areindependent of one another.

[0025] 1. Morphology and colour of the colonies: short-branched hyphae,which disintegrate into rod- and spore-like elements. The colonies ofGF270 and GF376 are salmon-pink (RAL 3022) and those of GF578 and GF674are light red (RAL 3012).

[0026] 2. Diamino acids of the peptidoglycan: meso-diaminopimelic acid

[0027] 3. Mycolic acids: Rhodococcus mycolic acids: The determination ofthe long-chain mycolic acid was carried out by means of high-temperaturegas chromatography. The elution profiles of the mycolic acids of GF270and GF376 and those of GF473, GF578 and GF674 were identical. Themycolic acid length for GF270 and GF376 was C₃₈-C₄₆ and that for GF473,GF578 and GF674 was C₄₀-C₄₈. The mycolic acid patterns were comparedwith mycolic acid patterns of Rhodococcus strains. GF270 was identifiedwith a very low correlation factor (0.086) as belonging to Rhodococcusrhodochrous; it was not possible to identify GF376 by this method. Theother three isolates GF473, GF578 and GF674 were identified with a verylow correlation factor as belonging to Rhodococcus ruber.

[0028] 4. Fatty acid pattern: unbranched, saturated and unsaturatedfatty acids including tuberculostearic acid. The fatty acid pattern isdiagnostic of all Rhodococcus genera and closely related Mycobacterium,Nocardia, Dietzia, Tsukamurella and some Corynebacteria species. Theidentification at the species level was obtained by qualitative andquantitative differences in the fatty acid pattern of GF270, GF376,GF473, GF578 and GF674 with the fatty acid patterns of Rhodococcusspecies.

[0029] 5. The 16S rDNA subsequences of GF270 and GF376 were identical(100%), although the comparison of them with the Rhodococcus strainsonly showed 99.1% similarity to the closest related Rhodococcusrhodochrous. GF473 and GF578 were identical in their 16S rDNA sequence(100%). GF674 differs from GF578 in only one base pair of 500 (99.8%).All three isolates show only a distant relationship with Rhodococcuscoprophilus (98.4%).

[0030] Based on the chemotaxic and molecular biology results, it can beconcluded that GF270 and GF376 on the one hand and GF473, GF578 andGF674 on the other hand are strains of 2 new Rhodococcus species. GF270and GF376 are closely related to Rhodococcus rhodochrous in their 16SrDNA (99.1%), however GF473, GF578 and GF674 are only distantly relatedto Rhodococcus coprophilus (98.4%).

[0031] The enzyme extract can be obtained by professionally customarydisruption of the microorganisms, such as, for example, by disruption bymeans of ultrasound, by means of the French press method or the lysozymemethod. This enzyme extract and, of course, also the completemicroorganism cells can be immobilized on a suitable support material,customarily embedded in a polymer, for carrying out the process, orabsorbed on a suitable support material.

[0032] The enzymes according to the invention having nitrile hydrataseactivity are obtainable from the microorganisms of the genusAmycolatopsis and are able to convert a nitrile into an amide, inparticular they are obtainable from Amycolatopsis NA40 (DSMZ 11617).

[0033] These enzymes in particular have the following properties:

[0034] a) a pH optimum of pH 6.5±1.0

[0035] b) a temperature optimum between 35 and 40° C. at a pH of 7.0

[0036] c) a K_(M) value for the substrate 3-cyanopyridine of 41.7 mM±7.7mM (20° C., 45 mM phosphate buffer, pH 7.0)

[0037] in particular the enzymes have a

[0038] d) molecular weight of 106 kDa, such as, for example, determinedby SDS-PAGE.

[0039] Nitriles can generally be employed as substrates for thebiotransformation. Expediently, either aliphatic nitriles having 1 to 10carbon atoms, optionally substituted by, for example, hydroxyl, amino,halogen or carboxyl, or substituted or unsubstituted aromatic nitrileshaving 4 to 10 carbon atoms in the aromatic ring system are employed.Aliphatic nitriles having 1 to 10 carbon atoms which can be used aredinitriles, hydroxynitriles, aminonitriles such as, for example,n-octanenitrile, cyanoacetic acid, isocapronitrile, n-valeronitrile,adiponitrile, glutaronitrile, succinonitrile, sebaconitrile,propionitrile, crotononitrile, acrylonitrile, methacrylonitrile,n-butyronitrile or azelanitrile. Aromatic nitriles having 4 to 10 carbonatoms which can be used are nitriles of the general formula

[0040] in which R¹ and R² are a hydrogen atom, a halogen atom orC₁₋₄-alkyl. F, Cl, Br or I can be used as halogen atom. Methyl, ethyl,propyl, isopropyl, tert-propyl, butyl, isobutyl or tert-butyl can beused as C₁₋₄-alkyl. Expedient representatives of the aromatic nitrilesof the general formula I or II are 2-, 3- or 4-cyanopyridine,benzonitrile, fluoro-, chloro- or bromobenzonitrile, such as, forexample, o-, m- or p-chlorobenzonitrile or 2-chloro-3-cyanopyridine.3-Cyanopyridine is preferably used as aromatic nitrile having 4 to 10carbon atoms.

[0041] The biotransformation is expediently carried out with addition ofsubstrate in one portion or continuously such that the substrateconcentration does not exceed 40% by weight, preferably 30% by weight.

[0042] The process is expediently carried out with resting (non—growing)cells.

[0043] Suitable media for the biotransformation are those customary inthe specialist field, such as, for example, low molecular weightphosphate buffers, HEPES buffers, citrate buffers, borate buffers, themedia according to Tables 1 to 3 or modified forms thereof such as, forexample, those described in Example 8 (1) or TRIS/HCl buffers.

[0044] The biotransformation is expediently carried out at a temperaturefrom 0 to 50° C. and at a pH between pH 4.5 and pH 10, preferably at atemperature from 20 to 40° C. and at a pH between pH 4.5 and pH 10.0.

[0045] In a particularly preferred embodiment, the biotransformation canbe carried out in the presence of C₁₋₄-alcohols. C₁₋₄-alcohols employedcan be methanol, ethanol, propanol or butanol. Methanol is preferablyused.

[0046] After the reaction, the corresponding amides can then be isolatedby customary working-up methods such as, for example, bycrystallization.

EXAMPLES Example 1

[0047] Growth of Microorganisms of the Genus Actinomadura orAmycolatopsis

[0048] a) Various soil samples were inoculated with various nitriles oramides as a C and N source in the enrichment medium according to Table 1and incubated at 37° C. or 45° C. for 7-10 days. The cultures were thentransferred to the same medium and again cultured at 37° C. for 7-10days. The whole process was repeated 3 times. The cultures were thendiluted and plated out in order to obtain individual colonies. Theplates were incubated at 37° C. for 5 days. The different colonies werethen tested for the desired activity.

[0049]  Amycolatopsis NA40 (DSMZ 11617) and Amycolatopsis NE31 (DSMZ11616) were isolated in this way and then grown in the optimized medium(Table 3) for 90-100 h with shaking at 37° C.

[0050]  Adiponitrile served as a C and N source for Amycolatopsis NE31(DSMZ 11616), Actinomadura spadix E3733 and Actinomadura spadix E3736,azelanitrile served as a C and N source for Amycolatopsis NA40 (DSMZ11617) and Actinomadura spadix 45A32, n-octanenitrile served as a C andN source for Actinomadura spadix 4501 and cyanoacetic acid served as a Cand N source for Actinomadura spadix C15.

[0051] b) Amycolatopsis NA40 was cultured in the medium according toTable 3. The culturing was carried out for 2 or 3 to 4 days at atemperature of 37° C. under aerobic conditions in subcultures (4ml/tube) and in a “main culture” (500 ml/flask). The cell growth wasmeasured turbidimetrically at 610 nm and the dry weight of the cells wascalculated in the following way: weight of the dry cells inmg/ml=OD_(610 nm) ×0.277. TABLE 1 Enrichment medium Nitrile 2.0 g KH₂PO₄7.0 g MgSO₄.7H₂O 0.1 g Vitamin mixture 1.0 ml CoCl₂.6H₂0 2.0 mgFeS0₄.7H₂0 2.0 mg

[0052] TABLE 2 Basal medium Maltose 2.0 g NaNO₃ 1.0 g K₂HPO₄ 0.1 gMgSO₄.7H₂0 0.05 g 

[0053] TABLE 3 Optimized medium D-glucose 4.5 g Meat extract 0.5 gK₂HPO₄ 0.1 g MgSO₄.7H₂O 0.05 g CoCl₂.6H₂O 1.0 mg

Example 2

[0054] Biotransformations with Microorganisms of the Genus Actinomaduraor Amycolatopsis

[0055] (1) For determination of the nitrile hydratase activity, areaction mixture (2 ml) containing 3-cyanopyridine (1.0 M, 1.0 ml),potassium phosphate buffer (pH 7.0, 0.1 M, 0.5 ml) and 0.5 ml of cellsuspension were incubated at 20° C. for 30 min with stirring. Thereaction was stopped by addition of 0.2 ml of 3 N HCl. Aftercentrifuging briefly, the nicotinamide formed was determined by means ofHPLC (Shimadzu SPD 6A system using a C18 column (Develosil ODS-HG-5,4.6×250 cm); eluent: 10 mM KH₂PO₄/H₃PO₄ (pH 2.8)/acetonitrile 9:1 (v/v);flow rate: 1 ml/min; the absorption was measured at 230 nm). Thespecific activity was expressed as μmol of nicotinamideformed/ml/min/OD_(610 nm).

[0056]  The reaction rates of aliphatic nitriles in the enrichmentmedium (Table 1) with isolated bacteria is summarized in Table 5, theeffects of inducers and cofactors in the basal medium (Table 2) issummarized in Table 4 and the activity comparison of Amycolatopsis toRhodococcus in the basal medium (Table 2) is summarized in Table 6. Theresults from Table 4 show that the nitrile hydratase from AmycolatopsisNA40 is constitutively expressed but the cofactor cobalt is necessaryfor the activity.

[0057] (2) Effect of the temperature on the growth of NA40

[0058]  Subcultures (2 ml) were incubated at 37° C. for 2 days in themedium according to Table 3, and then transferred to shaker flaskscontaining 20 ml of medium according to Table 3. Culturing was carriedout at 37, 40, 45, 50 and 55° C. for 3 to 4 days with shaking. The cellgrowth was measured and the nitrile hydratase activity was determined at20° C. Table 7 shows the effect of the temperature on the nitrilehydratase activity and on the cell growth. TABLE 4 Effects of Inducersand cofactors on the specific activity in the basal medium TotalSpecific activity activity Growth (μmol/ml/ (μmol/ml/ (OD_(610 nm)) min)min/OD) Inducer — 1.26 20.9 16.6 ε-Capro- 0.66 9.52 14.5 lactamCrotonamide 3.41 22.9 6.72 Meth- 3.33 2.46 0.74 acrylamide Butyramide2.19 0.19 0.88 Propionamide 1.91 0.92 0.48 Urea 1.72 2.97 1.73 Cofactor— 7.97 0.10 0.01 FeSO₄.7H₂O 8.32 3.36 0.40 CoCl₂.6H₂O 8.41 47.8 5.68

[0059] TABLE 5 Conversion rates of aliphatic nitriles using the isolatedbacteria Growth Total activity Specific activity Strains Substrates(OD_(610 mm)) (μmol/ml/min) (μmol/ml/min/OD) Amycolatopsis NE31 (DSMZAdiponitrile 2.68 0.377 0.141 11616) Actinomadura E3733 Adiponitrile1.62 0.347 0.214 spadix Actinomadura E3736 Adiponitrile 1.36 3.00 2.21spadix Actinomadura 45A32 Azelanitrile 5.81 18.8 3.23 spadixActinomadura 45O1 n-octane- 7.24 32.2 4.45 spadix nitrile ActinomaduraC15 Cyanoacetic 2.04 7.01 3.43 spadix acid Amycolatopsis NA40 (DSMZAzelanitrile 5.92 33.0 5.57 11617)

[0060] TABLE 6 Activity of Amycolatopsis in comparison to Rhodococcusrhodochrous J1 Microorganism Amycolatopsis Purified enzyme Purifiedenzyme NA40 Microorganism from NA40 from J1 (DSMZ 11617) Rhodococcus(μmol/min/mg (μmol/min/mg (μmol/ml/min ) rhodochrous J1 protein)protein) Activity for 303 314 1110 371 3-cyanopyridine

[0061] (3) For determination of the activity of NA40 with respect to anumber of substrates, cells having a dry weight of 0.0388 mg wereincubated in the buffer described above. The reaction was started byaddition of the appropriate substrate and incubated at 20° C. withshaking for 10 min. The reaction was stopped by addition of 0.2 ml of 2N HCl and the reaction mixture was briefly centrifuged. The supernatantwas analysed by means of HPLC or gas chromatography. Table 8 shows thetest conditions for the substrate specificity and Table 9 shows thesubstrate specificity of resting NA40 cells for various substrates.

[0062]  The respective test conditions are summarized in Table 8 and theresults are summarized in Table 9. TABLE 7 Effect of the growthtemperature on the nitrile hydratase activity and on the cell growthSpecific Total activity activity (μmol/ Relative Tempera- Growth (μmol/ml/min/ activity ture (mg/ml) ml/min) mg) (%) 37° C. 6.16 4.69 0.761 10040° C. 5.79 9.89 1.71 225 45° C. 6.56 4.83 0.736 97 50° C. 5.96 1.160.195 26

[0063] TABLE 8 Test conditions for substrate specificity SubstrateAnalysis Substrate (mM) method Amide formed 3-Cyanopyridine 1.0 HPLCNicotinamide 2-Cyanopyridine 0.25 HPLC 2-Picolinamide 4-Cyanopyridine0.25 HPLC Pyridine- 4-carboxamide Crotononitrile 0.4 HPLC CrotonamideBenzonitrile 0.03 HPLC Benzamide Acrylonitrile 0.4 HPLC Acrylamideo-Chlorobenzo- 0.15 HPLC o-Chlorobenz- nitrile amide m-Chlorobenzo- 0.15HPLC m-Chlorobenz- nitrile amide p-Chlorobenzo- 0.15 HPLC p-Chlorobenz-nitrile amide 2-Chloro- 0.15 HPLC 2-Chloro- 3-Cyanopyridine nicotinamideAcetonitrile 0.4 GC Acetamide Propionitrile 0.4 GC PropionamideMethacrylo- 0.4 GC Methacrylamide nitrile n-Butyronitrile 0.4 GCn-Butyramide

[0064] TABLE 9 Substrate specificity of NA40 nitrile hydratase Relativeactivity Substrate (%) 3-Cyanopyridine 100 4-Cyanopyridine 1682-Cyanopyridine 128 Benzonitrile 51 Crotononitrile 52 Acrylonitrile 115o-Chlorobenzo- 96 nitrile m-Chlorobenzo- 75 nitrile p-Chlorobenzo- 16nitrile 2-Chloro- 126 3-cyanopyridine Acetonitrile — Propionitrile 105Methacrylo- 130 nitrile n-Butyronitrile 194

[0065] (4) Temperature optimum and thermal stability in resting cells

[0066]  The reaction was carried out in the standard reaction mixturefor 10 min. The temperature optimum was between 35 and 40° C. (FIG. 5).The cells were then incubated at various temperatures for 30 min and theactivity was tested under standard reaction conditions. As can be seenfrom FIG. 4, the heat stability was 40° C.

[0067] (5) pH Optimum and pH stability in resting cells

[0068]  For this purpose, the reaction was carried out for 10 min in thestandard reaction mixture in which the potassium phosphate buffer hadbeen replaced by various 0.1 M buffers. As can be seen from FIG. 6, thepH optimum was between 4.5 and 10. After the cell suspension had beenincubated at 20° C. for 30 min at various pHs, the cells werecentrifuged. The cells were then washed and resuspended in 0.1 Mpotassium phosphate buffer pH 7.0. The reaction was carried out for 10min by addition of 3-cyanopyridine under standard conditions. The enzymewas stable between pH 4.5 and pH 10.0 (FIG. 7).

[0069] (6) Accumulation of nicotinamide from 3-cyanopyridine by means ofNA40

[0070]  The reaction was carried out in a reaction mixture (30 ml),comprising 500 mM 3-cyanopyridine, 40 mM potassium phosphate buffer (pH7.0) and resting cells (dry weight 2.3 mg). During the reaction,3-cyanopyridine (500 mM) was added 7 times as soon as it was consumed.In this manner, 4.0 M 3-cyanopyridine was added in the course of 15 hand 3.89 M (475 g/l) nicotinamide was formed, corresponding to a yieldof 97.3%. Nicotinic acid was not formed.

Example 3

[0071] Identification of Microorganisms of the Genus Amycolatopsis

[0072] The following 5 chemotaxonomic markers supported theidentification:

[0073] 1. Diagnostic amino acid of the peptidoglycan: mesodiaminopimelicacid

[0074] 2. Diagnostic sugars: arabinose and galactose

[0075] 3. Mycolic acids: mycolic acids absent

[0076] 4. Menaquinones: MK-9 (H₄)

[0077] 5. Fatty acid pattern: iso/anteiso-branched and 2-hydroxy fattyacids, small amounts of 10-methyl-branched fatty acids were additionallydetected. This fatty acid pattern was found in all representatives ofthe genus Amycolatopsis (fatty acid pattern 3f)

[0078] The combination of these chemical features is diagnostic of allspecies of the genus Amycolatopsis.

[0079] The fatty acid data of the two cultures were compared with theaid of main component analyses using the entries in the fatty aciddatabase. Using this method, it was possible to assign both NE31 andNA40 to the genus Amycolatopsis, an identification of the species,however, was not possible, since the correlation factor was too low. Thecomparison of the fatty acid patterns of both strains showed, however,that they are two strains of different types.

[0080] The result was confirmed by the results of the 16S rDNA sequenceanalysis.) Here too, assignment to the genus Amycolatopsis took place,but not to any of the Amycolatopsis species described. In this method,the sequence of the 16S rDNA was determined by the direct sequencing ofthe PCR-amplified 16S rDNA gene. The diagnostic part of the 16S rDNAsequence was compared with the sequences of the type species of thegenus Amycolatopsis and related taxa. The result showed that the strainbelongs to the genus Amycolatopsis. The highest agreement was found toAmycolatopsis methanolica at 96.9% (NA40) and 96.1% (NE31). Betweenthem, the two isolates showed agreement in the sequences of 99.0%. Ourinvestigations on representatives of the genus Amycolatopsis have shownthat for a good species identification the correlation factor must behigher than 99.5%. Since at 96.9% the value is clearly below 99.5%, itcan be assumed from this that the two isolates were not representativesof known Amycolatopsis species.

[0081] On the basis of the present results, it was not possible toassign the isolates to any of the known Amycolatopsis species. We assumefrom this that NA40 and NE31 are strains of two new, previouslyundescribed species of the genus Amycolatopsis.

[0082] Identification Characteristics of Microorganisms of the GenusAmycolatopsis

[0083] Colour of aerial mycelium

[0084] Colour of substrate mycelium

[0085] Colour of diffused pigment Sugar spectrum ARA + GAL + MAD − XYL −GLU V RIB + Type A DAP DL Menaquinones (in %) 8/4 − 9/0 (+) 9/2 + 9/4+++ 9/6 − 9/8 − 16S rDNA homology >99.5% Phospholipids PE + OH-PE + lysoPE − met PE − PC − NPG − PI + PIM V PG + DPG + GL − Type II + OH-PEFatty acids iso 16 +++ iso 15 + iso 17 (+) anteiso 15 + anteiso 17 (+)10-Me 16 (+) 10-Me 17 + 2-OH 15 + 2-OH 16 + Type 3f MS −

Example 4

[0086] Purification of the Nitrile Hydratase from Microorganism StrainNA40

[0087] The strain was cultured at 37° C. for 3 days in the mediumaccording to Table 3. The cells of a 2 l culture were harvested by meansof centrifugation and then resuspended in 0.85% strength NaCl solution.The cells were then transferred to 0.1 M potassium phosphate buffer (pH7.0) comprising 44 mM n-butyric acid and treated with ultrasound. Thecell extract was centrifuged and the cell fragments were removed. Thisextract was used for the enzyme purification.

[0088] During the entire purification, potassium phosphate buffer (pH7.0) comprising 44 mM n-butyric acid was used. As can be seen from Table10, the enzyme was purified to homogeneity in 3 steps. TABLE 10Purification of the nitrile hydratase from NA40 Total Total Specificactivity protein activity Enrich- (Units) (mg) (U/mg) ment Cell-free73,300 1020 71.9 1 extract DEAE- 68,000 110 620 8.62 Sephacel Phenyl-64,800 61.4 1105 15.4 TOYOPEARL

Example 5

[0089] Characterization of the Nitrile Hydratase

[0090] (1) Determination of the molecular weight, the subunit structureand the cobalt ion content

[0091]  The molecular weight was determined to be 106 kDa bychromatography on a TSK gel column G3000 SW (0.75×60 cm) using a 0.1 Mpotassium phosphate buffer (pH 7.0) containing 0.2 M KC1 and 44 mMn-butyric acid. It was determined that the enzyme consists of 2different subunits α and β, whose molecular weight was determined to be30,000 and 26,000 in each case.

[0092]  FIG. 1 shows the determination of the molecular weight bychromatography on TSK gel G3000 SW.

[0093]  FIG. 2 shows the determination of the molecular weight by meansof SDS-PAGE

[0094]  FIG. 3 shows the absorption spectrum of the purified enzyme. Abroad absorption of 300-400 nm was observed.

[0095] (2) Substrate specificity of the purified enzyme

[0096]  The substrate specificity was determined analogously to Example2 (1). The results are summarized in Table 11. TABLE 11 Substratespecificity of the purified nitrile hydratase Relative activity (%)Total Reaction activity with (μmol/ Enzyme resting Substrate (M) ml/min)reaction cells 3-Cyanopyridine 1.0 17.7 100 100 2-Cyanopyridine 0.2539.1 221 128 4-Cyanopyridine 0.25 31.6 179 168 Crotononitrile 0.4 11.967 52 Benzonitrile 0.03 11.3 64 51 Acrylonitrile 0.4 16.6 94 115o-Chlorobenzo- 0.15 22.4 127 96 nitrile m-Chlorobenzo- 0.15 15.9 90 75nitrile p-Chlorobenzo- 0.15 2.30 13 16 nitrile 2-Chloro- 0.15 16.0 90126 3-Cyanopyridine Acetonitrile 0.4 — — — Propionitrile 0.4 39.3 222105 Methacrylo- 0.4 22.1 125 130 nitrile n-Butyronitrile 0.4 17.9 101194

[0097] 1.7 Units of enzyme were added to the reaction mixture (2.0 ml).The reaction mixture contained the respective substrate in 45 mMphosphate buffer (pH 7.0).

[0098] (3) Determination of the K_(M) value

[0099]  The K_(M) value was determined to be 41.7 mM for 3-cyanopyridineand to be 3.7 mM for acrylonitrile by means of the Lineweaver-Burkdiagram. Compared with Rhodococcus rhodochrous J1, which had a K_(M)value relative to 3-cyanopyridine of 200 mM, that of NA40 issignificantly lower. This is one of the main advantages of NA40.

[0100] (4) Heat stability and temperature optimum

[0101]  The purified enzyme was incubated for 30 min at pH 7.0 atdifferent temperatures and the conversion of 3-cyanopyridine tonicotinamide was then measured at 20° C. for 1 min. The enzyme wasinactivated at a temperature of greater than 40° C. The heat stabilitywas about 40° C. as in resting cells and the temperature optimum wasbetween 35 and 40° C. (FIG. 5).

[0102] (5) pH Optimum and pH stability

[0103]  For this purpose, the conversion of 3-cyanopyridine tonicotinamide was carried out at 20° C. in a reaction mixture (2.0 ml)comprising various buffers (42.5 mM), 1.71 units of purified enzyme and500 mM 3-cyanopyridine. The pH optimum was at about pH 6.5±1.0 (FIG. 8).

[0104]  For determination of the pH stability, 4.2 units of purifiedenzyme were incubated at 20° C. for 1 h in various buffers (45 mM). Apart of the incubated solution, 1.71 units, were added to the standardreaction mixture (cf. Example 2(1)). The remaining activity wasdetermined. The enzyme was stable in a pH range from pH 5-9. The resultis shown is FIG. 9.

[0105] (6) Inhibitors

[0106]  The effect of various inhibitors was determined. The results aresummarized in Table 12. TABLE 12 Effect of various inhibitors on thepurified enzyme Relative Inhibitor mM activity (%) — 100N-ethylmaleimide 1 97 Iodoacetic acid 1 39 4-Chloromercurobenzoic acid0.1 69 Sodium azide 1 59 Hydroxylamine 1 37 Phenylhydrazine 1 8Semicarbazide 1 82 Tiron (disodium salt of 4,5-dihydroxy-1,3-benzene- 1110 disulphonic acid o-Phenanthroline 1 89 α,α′-Dipyridyl 1 1008-Hydroxyquinoline 1 110 EDTA (ethylenediaminetetraacetic acid 1 115Diethyl dithiocarbamate 1 89

Example 6

[0107] Effect of Methanol on Resting Cells of NA40

[0108] The reaction was carried out for 10 min in the presence of 0-20%(v/v) methanol according to Table 13. As shown in Table 14, the activityis increased by the addition of 5-15% methanol. TABLE 13 Reaction withresting cells Methods {circle over (1)} {circle over (2)} {circle over(3)} {circle over (4)} {circle over (5)} 1.0 M 1.0 ml 1.0 ml 1.0 ml 1.0ml 1.0 ml 3-cyano- pyridine 0.1 M KPB* 0.9 ml 0.8 ml 0.7 ml 0.6 ml 0.5ml (pH 7.0) Methanol — 0.1 ml 0.2 ml 0.3 ml 0.4 ml Cell 0.1 ml 0.1 ml0.1 ml 0.1 ml 0.1 ml suspension (0.388 mg/ ml) Total volume 2.0 ml 2.0ml 2.0 ml 2.0 ml 2.0 ml

[0109] TABLE 14 Effect of methanol on Amycolatopsis NA40 MethanolRelative activity Methods [% (v/v)] [%] {circle over (1)} 0 100 {circleover (2)} 5 123 {circle over (3)} 10 128 {circle over (4)} 15 130{circle over (5)} 20 105

Example 7

[0110] Enrichment of Microorganisms of the Genus Rhodococcus

[0111] Various soil samples were inoculated with cyanoacetic acid as a Cand N source in the enrichment medium according to Table 1 and themicroorganisms Rhodococcus GF270, GF578, GF473 and GF376 were isolatedaccording to Example 1.

Example 8

[0112] Biotransformation using Microorganisms of the Genus Rhodococcus

[0113] (1) Heat Stability of the Microorganisms Rhodococcus GF674,Rhodococcus GF578, Rhodococcus GF270 and Rhodococcus GF376 in comparisonwith Rhodococcus rhodochrous J1.

[0114]  For determination of the heat stability, the microorganismsdescribed above were cultured in the following media.

[0115]  Rhodococcus rhodochrous J1 was cultured for 72 h in the mediumdescribed in EP-A 0 307 926. The microorganisms Rhodococcus GF674,GF578, GF270 and GF376 were cultured in the following media at pH 7.0for up to 96 h:

[0116]  Rhodococcus GF674 in a medium comprising yeast extract 1.0 g/l,fructose 5.0 g/l, malt extract 10.0 g/l, acetamide 5.0 g/l, KH₂PO₄ 2.0g/l, MgSO₄. 7H₂O 0.5 g/l and CoCl₂. 6H₂O 10.0 mg. Rhodococcus GF578 in amedium containing yeast extract 1.0 g/l, fructose 15.0 g/l, malt extract10.0 g/l, acetamide 25.0 g/l, KH₂PO₄ 2.0 g/l, MgSO₄. 7H₂O 0.5 g/l andCoCl₂. 6H₂O 5.0 mg. Rhodococcus GF270 in a medium containing yeastextract 12.5 g/l, sodium citrate 5.0 g/l, methacrylamide 7.5 g/l, KH₂PO₄2.0 g/l, MgSO₄. 7H₂O 0.5 g/l and CoCl₂. 6H₂O 30.0 mg. Rhodococcus GF376in a medium containing yeast extract 1.0 g/l, sodium citrate 10.0 g/l,malt extract 15.0 g/l, butyramide 7.5 g/l, KH₂PO₄ 2.0 g/l, MgSO₄. 7H₂O0.5 g/l and CoCl₂. 6H₂O 15.0 mg.

[0117]  The resting cells were then incubated for 15 min at varioustemperatures and the remaining activity was then determined under thestandard reaction conditions according to Example 2(1).

[0118]  In the course of this it was found that Rhodococcus GF674 had arelative activity of nearly 100% at a temperature of 50° C. and stillapproximately an activity of 10% at 60° C. Rhodococcus GF578 likewisehad 100% relative activity at 50° C. and a relative activity of 20% at60° C. Rhodococcus GF376 had 100% relative activity up to 50° C., 70%relative activity at 60° C. and nearly still 5% relative activity at 70°C. Rhodococcus GF270 had a relative activity of nearly 100% up to 60° C.and likewise still 5% relative activity at 70° C. In comparison to this,Rhodococcus rhodochrous J1 had 100% relative activity up to 50° C., 80%at 60° C. and no longer any activity at 70° C.

[0119]  In summary, it can therefore be stressed that Rhodococcus GF270and GF376 had a better heat stability than J1 and GF270 had the bestheat stability.

[0120] (2) pH Optimum of the Rhodococcus strains

[0121]  The effect of the pH on the nitrile hydratase activity of theRhodococcus strains GF674, GF578, GF270 and GF376 was determined asdescribed in Example 2(5).

[0122]  The pH optimum of Rhodococcus GF674 was at pH 7.5-9.5, of GF578at pH 8-8.5, for GF270 at pH 6-7.0 and for GF376 at pH 6-8.

[0123] (3) Substrate specificity of the Rhodococcus strains

[0124]  The substrate specificity is summarized as relative activity inTable 15.

[0125] (4) Nicotinamide accumulation of the Rhodococcus strains

[0126]  Analogously to Example 2(6), the Rhodococcus strains GF674,GF578, GF270 and GF376 were cultured with 3-cyanopyridine (about 500mM). In the course of this Rhodococcus GF674 formed 6 M nicotinamidewithin 25 h, GF578 5.5 M nicotinamide within 10 h, GF270 about 8.5 Mnicotinamide within 20 h and GF376 7.5 M nicotinamide within 20 h.

[0127] (5) Tolerance of 3-cyanopyridine on the activity of theRhodococcus strains

[0128]  In order to test the tolerance of 3-cyanopyridine, resting cellswere incubated for 15 min at 20° C. at concentrations of 3-cyanopyridinebetween 1 and 10% (w/v) and the cells were then removed bycentrifugation. After washing the cells with 0.85% NaCl, the remainingactivity was measured.

[0129]  The tolerance of 3-cyanopyridine as a substrate was tested atvarious substrate concentrations. It was found that at a substrateconcentration of 2% (w/v) the nitrile hydratase activity of Rhodococcusrhodochrous J1 decreased by the factor 1.4, the nitrile hydrataseactivity of Rhodococcus GF674 at a substrate concentration of 4% (w/v)decreased by the factor 1.4, the nitrile hydratase activity ofRhodococcus GF578 remained nearly constant up to a substrateconcentration of 8%, the nitrile hydratase activity of Rhodococcus GF270at a substrate concentration of 4% (w/v) decreased by the factor 1.17and the nitrile hydratase activity of Rhodococcus GF376 at a substrateconcentration of 10% (w/v) decreased by the factor 1.25.

[0130]  In comparison with the other Rhodococcus strains, Rhodococcusrhodochrous Ji exhibited the poorest tolerance for 3-cyanopyridine.TABLE 15 Rhodococcus rhodochrous Rhodococcus Rhodococcus RhodococcusRhodococcus J1 GF674 GF578 GF270 GF376 3-Cyanopyridine 100 (%) 100 (%)100 (%) 100 (%) 100 (%) 2-Cyanopyridine 45 308 200 15.7 54.34-Cyanopyridine 70 231 167 55.6 79.8 Benzonitrile 27 138 85.1 13.4 57.22-Chlorobenzonitrile 2.8 64.8 49.0 0 0 3-Chlorobenzonitrile 43 27.8 28.98.41 8.63 4-Chlorobenzonitrile 13 0 0 0 0 Acetonitrile 608 1.49 347 659806 Propionitrile 434 274 132 37.3 245 n-Butyronitrile 352 491 368 181195 Acrylonitrile 478 147 101 192 257 Crotononitrile 78.2 98.0 124 37.1110 Methacrylonitrile 86.9 176 122 0 0

1. Microorganisms of the genus Amycolatopsis or Actinomadura,characterized in that they are capable of converting a nitrile into anamide, and enzyme extracts thereof.
 2. Microorganisms according to claim1 of the species Amycolatopsis NA40 and Amycolatopsis NE31, for exampleas deposited under the deposit numbers DSM 11617 and DSM 11616, andtheir functionally equivalent variants and mutants.
 3. Microorganisms ofthe species Rhodococcus GF270 and GF376, for example as deposited underthe deposit numbers DSM 12211 and DSM 12175, and their functionallyequivalent variants and mutants, characterized in that they are capableof converting a nitrile into an amide, and enzyme extracts thereof. 4.Enzyme having nitrile hydratase activity, obtainable from themicroorganisms according to claims 1 and
 2. 5. Enzyme according to claim4, characterized by a) a pH optimum of pH 6.5±1.0 b) a temperatureoptimum between 35 and 40° C. at a pH of 7.0 c) a K_(M) value for thesubstrate 3-cyanopyridine of 41.7 mM±7.7 mM.
 6. Process for preparationof amides, characterized in that a nitrile, as substrate, is convertedto the corresponding amide by means of the microorganisms according toone of claims 1 to 3, using an enzyme extract of these microorganisms orby means of the enzyme according to claim
 4. 7. Process according toclaim 6, characterized in that the nitrile employed is an optionallysubstituted aliphatic nitrile having 1 to 10 carbon atoms.
 8. Processaccording to claim 6, characterized in that the nitrile employed is anoptionally substituted aromatic nitrile having 4 to 10 carbon atoms inthe aromatic ring system.
 9. Process according to claim 8, characterizedin that the aromatic nitrile is selected from the compounds of thegeneral formula

in which R¹ and R² are a hydrogen atom, a halogen atom or C₁₋₄-alkyl.10. Process according to one of claims 6 to 9, characterized in that thereaction is carried out at a temperature from 0 to 50° C. and at a pHfrom 4.5 to
 10. 11. Process according to one of claims 6 to 10,characterized in that the reaction is carried out by means ofmicroorganisms of the genus Amycolatopsis having the designation NA40(DSMZ 11617) or NE31 (DSMZ 11616) or using their functionally equivalentvariants and mutants.